7. PROJECT SUMMARY/ABSTRACT The overall goal of this research project is to develop a vascularized full thickness skin substitute, engraftment of which can provide a definitive clinical cure of chronic cutaneous ulcers, an unmet medical need. This application is a competitive Revision to add a specific aim to a funded R01 grant (HL-085416 ?Optimizing Therapeutic Revascularization by Endothelial Cell Transplantation?) submitted in response to RFA-HL-17-029 (Revision Applications for Regenerative Medicine Innovation Projects). The parent R01 grant is focused on establishing perfusion of tissue engineered grafts through the self-assembly of incorporated human endothelial cells (EC) into microvessels. We have found that the best source of EC for this purpose is the differentiated progeny of human endothelial colony forming cells (HECFC), an ?adult? stem cell type isolated from neonatal cord or adult peripheral blood as defined by the terms of this RFA. Over the past six months, Drs. W. Mark Saltzman and Jordan S. Pober, co-PIs of the parent grant, have established a collaboration with Dr. Pankaj Karande to apply their approach for microvessel generation to his approach for 3D printing to create a full thickness human skin substitute. Current FDA-approved skin substitutes have all had only limited clinical success because they fail to vascularize, and consequently slough over the course of a few weeks. The vascularized skin substitute produced through this collaboration can potentially address this problem. However, the skin substitutes that we have generated to date employ bio-inks containing human cells that have been cultured in fetal bovine serum and use extracellular matrix molecules derived from animal sources. Similar exposures to animal proteins used in the production of currently approved skin substitutes have further limited their approved use. The goal of our new specific aim is to develop a vascularized full thickness human skin substitute through 3D printing made with bio-inks that are composed of human cells that have not been exposed to animal proteins and extracellular matrix molecules that are either derived from human tissues or made by recombinant approaches that do not involve exposure to animal proteins. Subaims a and b describe our approaches to achieve this. Subaim c will involve evaluation of perfusion of the resultant 3D bioprinted skin graft compared to natural skin and to Apligraf, a currently approved full thickness skin substitute, after engraftment on immunodeficient mouse hosts. The development of methods for creating a fully humanized and vascularized full thickness skin equivalent will provide the information needed to develop Good Manufacturing Practice (GMP) conditions for preparing clinical grade skin substitutes.